Phytic acid or phytate (myo-inositol hexakisphosphate) is the primary storage form of phosphorus in most plants and is abundant in seeds and legumes. However, the monogastric animals cannot utilize phosphorous from phytate due to the lack of necessary enzymes in digestive tract. Supplementation of inorganic phosphates was used to compensate the shortage in phosphorus ingestion but the excessive phosphorus in animal excretion has caused environmental pollution. In addition, the insoluble complexes formed by the highly negatively charged phytate with proteins and metal ions are major anti-nutritional factors. Phytase can hydrolyze phytate to lower inositol phosphates to release inorganic phosphate and thus has been widely applied in animal feeds to increase phosphorus availability and reduce phosphorus pollution. To date, phytase is estimated to account for 60% of feed enzyme products. Therefore, searching for phytases with higher specific activity and thermostability to lower the production cost and to survive the transient high-temperature step in pelleting procedure (80-85° C.) is of great interest to industries.
Classified by protein structure and catalytic property, there are four types of phytases including histidine acid phosphatases (HAPs), protein tyrosine phosphatase (PTP)-like phytases, purple acid phosphatases (PAPs) and β-propeller phytases (BPPs), with a majority of the characterized enzymes belonging to HAP. From previous studies, the crystal structures of all families except for PAP have been solved. Among the characterized phytases, Escherichia coli phytase (EcAppA), a member of the HAP family, has drawn much attention. First, EcAppA has high specific activity (up 56 to 2000 U/mg) under the favorable pH profile for feed additive. Second, large scale production of EcAppA in an industrial strain of Pichia pastoris has been successfully achieved by using fermentor for commercial applications. However, the need to enhance the enzymatic activity of EcAppA still remains.
Molecular engineering is a powerful approach to modify enzyme performances. Directed evolution involving random mutagenesis which builds a library provides a large pool of mutants for subsequent screening for useful mutants. But the efficiency is low and the procedure is laborious. A more ideal way is rational design, which is realized by the increasing information of protein structure and the development of powerful bioinformatics tools. Major obstacle in conducting a successful rational design is how to choose the useful residues or structural features.
In the present invention, site-directed mutagenesis of EcAppA is performed based on sequence comparison and structure analysis, so as to improve the industrial value of EcAppA.